Bladder cancer is one of the deadliest forms of cancer, considered the sixth most common cause of cancer-related death in the United States (Jemal et al., 2008). One of the major weapons in the arsenal of cancer fighting drugs is the use of the bacterium Mycobacterium bovis (Bacillus Calmette-Guerin, BCG) since 1976 to fight superficial urothelial carcinoma of the bladder (Herr and Morales, 2008; Kresowik and Griffith, 2010). For its use as an anticancer therapy, live BCG cells are taken from lyophilized powders and introduced into emptied bladders through a urethral catheter. After a residence varying from a few minutes to a few hours, the BCG cells are eliminated by the patients through emptying of the bladder. The patients are subsequently monitored by cystoscopy, conventional cytology and FISH analysis. The BCG effect is believed to be mediated through induction of an immune reaction in the bladder such as release of cytokines IL 8 and TRAIL that leads to tumoricidal activity (Herr and Morales, 2008; Kresowik and Griffith, 2010). This immune response is greatly amplified with repeated instillations of BCG, demonstrating the importance of elevated cytokine levels including IL2, IL6, IL8, TNF, and IFNs (Shintani et al., 2007; Bisiaux et al., 2009) and the subsequent infiltrations of neutrophils, lymphocytes, and monocytes/macrophages. Indeed, BCG-stimulated neutrophils have been shown to kill bladder cancer cells in vitro in a TRAIL-dependent manner (Ludwig et al., 2004).
Unfortunately, therapeutic use of live BCG cells for bladder cancer treatment is associated with many debilitating and/or serious side effects, ranging from cystitis and gross hematuria to life-threatening BCG sepsis. Such major side effects during intravesical BCG therapy can affect treatments in 30% of patients while mild cystitis, malaise, low grade fever and other side effects are common in about 90% of patients (Bohle et al., 2003; Sylvester et al., 2003).
Further, some recent observations endorses that certain single nucleotide polymorphisms (SNPs) in humans can promote disease progression in spite of BCG therapy (Basturk et al., 2006; Decobert et al., 2006), making use of the immune response-invoking live BCG bacteria less effective in such patients. Thus toxicity and efficacy issues have been a major deterrent in the use of live BCG cells in bladder cancer immunotherapy.
The ability of M. bovis BCG to attack cancer cells and suppress their growth is not unique to this organism. Indeed, many other bacteria such as Salmonella, Clostridia, Listeria, etc, are known to allow cancer regression both by inducing an immune response as well as actively growing in the core of the tumor (Mahfouz et al., 2007; Fialho and Chakrabarty, 2010a). Even many viruses have been designed for cancer therapy (Fialho and Chakrabarty, 2010a). Recently pathogenic bacteria such as Pseudomonas aeruginosa or gonococci/meningococci such as Neisseria meningitides reported to produce proteins such as azurin or Laz that demonstrate strong anticancer activity both in vivo and in vitro (Chakrabarty, 2010; Fialho and Chakrabarty 2010 a,b). Not only the full-length proteins, but peptides derived from them such as the 28 amino acid peptide P28 or the 26 amino acid peptide P26, derived from different parts of azurin, show entry specificity in cancer cells (Taylor et al., 2009) and high cytotoxicity in cancers such as breast, melanoma, prostate, brain, etc (Taylor et al., 2009, Chaudhari et al., 2007). Proteins such as azurin, considered a bacterial weapon against cancer (Chakrabarty, 2010; Fialho and Chakrabarty, 2010b), are secreted in response to the presence of cancer cells (Mahfouz et al., 2007), while the Neisserial protein weapon Laz is surface-exposed (Hong et al., 2006). The phase I human clinical trials of p28 were performed as per US-FDA guidelines. The p28 peptide has shown lack of toxicity and partial tumor regression in 2 patients and complete regression in 2 other patients out of 15 advanced stage (stage IV) cancer patients where no drugs were working and where the patients had less than 6 months life span. When given in 5 escalating doses 3 times a week for 4 weeks, followed by a break of 2 weeks, several patients showed stunted tumor growth but 2 patients showed partial regression and 2 patients showed complete regression of their tumors (where no drugs were working any longer). The 2 patients where the tumors completely regressed (making them disease free) as well as another patient (altogether 3) are alive today (middle of August 2011) beyond one and a half year (Richards et al., 2011), showing that such bacterial peptides as p28, derived from azurin, have unique modes of action so that they work against drug-resistant cancers. We hope that the newly identified peptide MB30 will behave similarly.
To the best of the knowledge of the applicant and/or inventors, no efforts have been made to look for protein/peptide weapons produced by such bacteria. However, considering the ability of microbial products being used as potential anticancer agent, the applicant/inventors carried out pains taking research to arrive at other protein weapons, either secreted or surface-associated in microorganisms including pathogens, or that could be designed as synthetic peptides, that would demonstrate anticancer activity for use as anticancer therapies instead of the live bacteria.